A Boronic Acid-Based Glutamine Analog Forms a Covalent Adduct with Kidney-Type Glutaminase and Suppresses...

Background: Cancer cells exhibit metabolic reprogramming characterized by increased dependence on glutamine to sustain rapid proliferation and biosynthetic demands. Kidney-type glutaminase (KGA), which catalyzes the first and rate-limiting step of glutamine metabolism, represents a promising therapeutic target, particularly in triple-negative breast cancer (TNBC), an aggressive sub-type lacking effective targeted therapies. This study evaluated 2-amino-4-boronobutyric acid (ABBA), a boronic acid-containing glutamine analog, as a potential KGA inhibitor with anticancer activity. Methods: KGA inhibition was assessed using a fluorometric enzymatic assay. Cytotoxic effects were examined in multiple TNBC cell lines. Covalent docking and molecular simulation analysis were performed to characterize interactions between ABBA and the KGA active site. Results: ABBA potently inhibited KGA activity, with an IC₅₀ of approximately 1.0 μM, demonstrating greater efficacy than several non-proteinogenic amino acid analogs. ABBA induced dose-dependent cytotoxicity across multiple TNBC cell lines, with pronounced sensitivity observed in basal sub-type cells and cellular sensitivity correlated with KGA expression levels. Expression of γ-glutamyl transpeptidase 1 (GGT1) was negligible, and, excluding any off-target effects, the observed anticancer effects are primarily attributed to KGA inhibition. Docking analysis indicated that ABBA forms a reversible covalent adduct with the catalytic Ser286 residue of KGA in a boronate tetrahedral geometry resembling transition-state mimics, while molecular simulation demonstrated stabilization of the complex through hydrogen bonding and electrostatic interactions. Conclusions: ABBA is a potent boron-based glutaminase inhibitor with therapeutic potential for targeting glutamine metabolism in TNBC. Further structural optimization and in vivo evaluation are warranted to advance ABBA toward therapeutic development.